One of the most striking features of virtually all normal tissues is the high degree of order and patterning that occurs during all stages of development. Whether one examines such diverse processes as the orderly formation of axon tracts, the creation of arrays of skeletal muscle fibers or the formation of kidney glomeruli, it is clear that the creation of normal tissue follows precise rules of organization. Little is known about how such order is generated in the body nor have techniques been developed that induce in a controllable manner the generation of multilayered ordered tissue structures in vitro or in vivo.
Just as order is a striking feature of normal tissues, so is disorder a feature of pathology. Disorder is seen in degenerative processes and is also seen in the failure of regeneration to create fully normal tissue. One common example of such disorder in tissue regeneration is seen in scar tissue in which the precisely patterned organization of cells that existed prior to injury is not reformed. When it occurs in deeper structures, function can be severely compromised. Indeed, the generation of ordered structures is so essential to the creation of a functioning nervous system that not even simple reflex loops can be established in its absence let alone the complexity of higher order motor and cognitive processes.
Since disorder is a feature of pathology and order is a feature of normal tissue function, and if normal tissue function requires the transmission of order through multiple layers of cells and the ability of cells to communicate to each other through transfer of factors, then it follows that a critical goal in the field of tissue repair or tissue engineering is the discovery of a means of creating structures that display order by allowing for the generation of multiple layers of cells and to enhance the ability of these cells to communicate with each other as a result of intentional design features utilized by the practitioner of the art.
While in general the field of tissue repair and tissue engineering has grown over the past decade to include various means for providing for new cell growth and new tissue formation, the area of nervous tissue regeneration has still proven to be perhaps the most complex. Nerve injuries complicate successful rehabilitation more than any other form of trauma. Painful neuroma formation, often more disabling than its associated sensory deficits, commonly causes major disability. Improvements in the techniques of nerve repair could provide better return of protective sensibility and tactile discrimination, reduce denervation atrophy of muscles, and prevent or minimize pain syndromes.
The nervous system is composed of numerous types of cells, including neurons and glial, or satellite cells. Glial cells include Schwann cells. The neurons carry signals between the brain and the rest of the body, while the primary role of the Schwann cells is to provide support for the neurons and to enhance the speed of electrical signals. Schwann cells also produce proteins essential for neuron growth (Bunge, M., (1994), J Neurol. December;242 (1 Suppl 1):S36-9; Tortora, (1992), Principles of Human Anatomy, Nervous System, 6th Edition, Chapter 16, Nervous Tissue, pp. 456-468). Each neuron is composed of a cell body, an axon, and dendrites. The tip of an axon is the growth cone and is responsible for navigation. Neurons can make multiple contacts with one or more neurons. The ability of neurons to extend neurites is of prime importance in establishing neuronal connections during development. It is also required during regeneration to re-establish connections destroyed as a result of a lesion. The organization of the contacts determines the overall function of the nervous system. The axons are surrounded by an insulating layer or myelin sheath formed by the Schwann cells (Tortora, (1992), Principles of Human Anatomy, Nervous System, 6th Edition, Chapter 16, Nervous Tissue, pp. 456-468). Injury to the axon that causes the Schwann cells to lose contact with the axons stimulates production of neurotrophic factors such as nerve growth factors. Nerve growth factor (NGF) has been shown to greatly enhance the growth of neurons in culture. With contact, regenerating axons stimulate Schwann cells to proliferate and form a basal lamina of collagen, proteoglycans, and laminin.
When a nerve is severed, a gap is formed between the proximal and distal portions of the injured nerve. In order for the nerve axon to regenerate and reestablish nerve function, it must navigate and bridge the gap. Under the appropriate conditions, e.g., minimal gap length, the proximal end forms neurite growth cones that navigate the gap and enter endoneural tubes on the distal portion. The growth cones sense the extracellular environment and determine the rate and direction of nerve growth. The motion of the axon is responsive to environmental signals provided by other cells that guide the growth cone (Tessier-Lavigne, (1994), Curr. Opin. Genet. Dev. August 4(4), pp. 596-601).
Once the growth cones reach the distal segment, they enter the endoneural tubes left from the degenerated axons. However, the growth cones and the dendrites on the proximal stump typically grow in many directions and unless the injury is small, the growth cones may never reach the distal segment. The natural ability of the nerve to regenerate is greatly reduced by the disruption of environmental cues resulting from, for example, soft tissue damage, formation of scar tissue, and disruption of the blood supply (Mackinnon and Dellon, (1988), J Hand Surg [Am]. November;13 (6):935-42; Fawcett and Keynes, (1990), Annu Rev Neurosci.;13:43-60, Buettner et al, (1994), Dev Biol., June;163(2):407-22).
Several techniques have previously been attempted to aid and accelerate the repair of damaged nerves: suturing the severed ends, suturing an allograft or autograft, or regenerating the nerve through a biological or synthetic conduit (Valentini et al., (1987), Exp Neurol., November; 98(2):350-6; Aebischer et al., (1988), Brain Res., June 28;454(1-2):179-87; Fenely et al., (1991), Exp Neurol., December; 114(3):275-85; Calder and Green, (1995) J Hand Surg [Br], August;20(4):423-8).
Autografts and allografts require a segment of a donor nerve acquired from the patient (autograft) or a donor (allograft). The donor nerve segment is removed from another part of the body or a donor and then sutured between the unattached ends at the injury site. Nerve autograft procedures are difficult, expensive, and offer limited success. Often, a second surgical procedure is required and may lead to permanent denervation at the nerve donor site. Allografts typically require the use of immunosuppressive drugs to avoid rejection of donor segments.
Artificial nerve grafts have been used in attempts to avoid the problems associated with autografts and allografts. Artificial grafts do not require nerve tissue from another part of the body or a donor. However, use of artificial nerve grafts has met with only limited success. Axonal regeneration in the peripheral nervous system has only been achieved for graft lengths up to approximately 3 cm in nonhuman primates. There has been little or no success with longer grafts. The previously used artificial nerve grafts were unsuitable for bridging longer gaps between distal and proximal nerve stumps and, therefore, are unsuitable for treating a significant proportion of nerve injuries.
Neurite growth has been aided to a limited extent by fabricating grooves on substrate surfaces (Clark et al., (1993), J Cell Sci., May;105 (Pt 1):203-12; Dow et al., (1991), Cell Tissue Res. August;265(2):345-51). The grooves employed in these studies were engraved on plastic or quartz substrates. The substrates utilized are unsuitable for implantation in vivo and thus the authors were unable to determine if the grooves could guide neurite growth in an animal. Alignment of neurons using physical guidance cues alone is highly uncertain and difficult to reproduce. For example, the neurites are typically aligned on only part of the substrate and unaligned on other parts and exhibit undesirable axon branching.
Tai et al., (Biotechnol. Prog. (1998), Vol. 14: 364-370) refer to the effects of micropatterned laminin glass surfaces on neurite outgrowth and growth cone morphology. In Tai et al., micropatterns consisting of laminin stripes alternating with glass stripes were formed on glass coverslips. Neuronal cultures were prepared from chicken dorsal root ganglia and seeded on either micropatterned laminin coverslips or on a uniform laminin coated glass surface. While neuronal growth on the micropatterned laminin surface was biased in the direction of the pattern, severe axon branching formed dense axon outgrowth. Thus, while the laminin provided some level of chemical guidance, applicability of the technique was limited. In addition, the glass substrates are unsuitable for implantation into patients.
More recently, tissue engineering approaches for nerve repair employ polymer conduits to protect the regenerating nerve and promote regrowth. Nerve conduits used for peripheral nerve and spinal cord injuries are typically termed guidance channels and bridges, respectively (G. R. Evans, Anat. Rec. 263 (4) (2001), 396-404; H. M. Geller, J. W. Fawcett, Exp. Neurol. 174 (2) (2002) 125-136). These conduits are implanted across the injury site and serve to support the damaged nerve by reducing infiltrating scar tissue, maintaining a continuous path, and directing axon outgrowth by physical guidance (C. E. Schmidt, J. B. Leach, Annu. Rev. Biomed. Eng. 5 (2003) 293-347). Conduits are typically fabricated with either single or multiple lumens (R. Talac, et al., Biomaterials 25 (9) (2004) 1505-1510; P. Aebischer, A. N. Salessiotis, S. R. Winn, J. Neurosci. Res. 23 (3) (1989) 282-289) and the lumen can be filled with a hydrogel (e.g. matrigel, fibrin) (R. O. Labrador, M. Buti, X. Navarro, Exp. Neurol. 149 (1) (1998) 243-252) or used empty (K. L. Gibson, J. K. Daniloff, G. M. Strain, Microsurgery 10 (2) (1989) 126-129). Single lumen conduits have been extensively used in peripheral nerve regeneration (V. B. Doolabh, M. C. Hertl, S. E. Mackinnon, Rev. Neurosci. 7 (1) (1996) 47-84). More recently, conduits with multiple, straight lumens have been proposed to segregate functional pathways, with each lumen acting as a guidance channel for axon growth (J. A. Friedman, et al., Neurosurgery 51 (3) (2002) 742-751 (discussion 751-752)). The material, mechanics, and physical properties of the conduits can affect the extent of nerve regeneration. Guidance channels have been fabricated from a range of natural and synthetic polymers (G. R. Evans, Anat. Rec. 263 (4) (2001) 396-404) using a variety of fabrication techniques, including solvent casting, extrusion, freeze drying, and dip molding (C. M. Patist, et al., Biomaterials 25 (9) (2004) 1569-1582; T. Hadlock, et al., Laryngoscope 109 (9) (1999) 1412-1416; M. S. Widmer, et al., Biomaterials 19 (21) (1998) 1945-1955). Materials used for fabrication include both natural (e.g., collagen) (M. Rafiuddin Ahmed, R. Jayakumar, Brain Res. 993 (1-2) (2003) 208-216; S. T. Li, et al., Clin. Mater. 9 (3-4) (1992) 195-200; S. J. Taylor, J. W. McDonald III, S. E. Sakiyama-Elbert, J. Control. Release 98 (2) (2004) 281-294) and synthetic polymers (e.g., silicone, ethylene vinyl coacetate (EVAc), poly(lactide-co-glycolide) (PLG)) (K. L. Gibson, J. K. Daniloff, G. M. Strain, Microsurgery 10 (2) (1989) 126-129; E. G. Fine, et al., Eur. J. Neurosci. 15 (4) (2002) 589-601). The processing of these materials can provide conduits with a range of degradation rates, porosities, and mechanical properties. Conduits with porosity ranging from semi-permeable to macroporous have been investigated, with the hypothesis that the porosity can allow access of soluble growth promoting factors or nutrients (M. S. Widmer, et al., Biomaterials 19 (21) (1998) 1945-1955; F. J. Rodriguez, et al., Biomaterials 20 (16) (1999) 1489-1500; C. B. Jenq, L. L. Jenq, R. E. Coggeshall, Exp. Neurol. 97 (3) (1987) 662-671; V. Maquet, et al., J. Biomed. Mater. Res. 52 (4) (2000) 639-651) from the surrounding environment. Additionally, the mechanical properties of the conduit must be sufficient to avoid channel collapse, which would limit neurite outgrowth and regeneration (C. E. Schmidt, J. B. Leach, Annu. Rev. Biomed. Eng. 5 (2003) 293-347; V. B. Doolabh, M. C. Hertl, S. E. Mackinnon, Rev. Neurosci. 7 (1) (1996) 47-84. In addition to providing structural support, nerve conduits can also function as a vehicle for localized delivery of neurotrophic factors (E. G. Fine, et al., Eur. J. Neurosci. 15 (4) (2002) 589-601; X. Xu, et al., Biomaterials 24 (13) (2003) 2405-2412). Neurotrophic factors (e.g., nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF)) are not typically produced in sufficient quantities after an injury (J. G. Boyd, T. Gordon, Mol. Neurobiol. 27 (3) (2003) 277-324). Localized delivery of these factors, which can be achieved by a pump, polymeric delivery, or the transplantation of engineered cells, has been employed to promote neuronal survival and stimulate neurite outgrowth following trauma (M. V. Chao, Nat. Rev., Neurosci. 4 (4) (2003) 299-309; S. David, S. Lacroix, Annu. Rev. Neurosci. 26 (2003) 411-440. The ability to combine neurotrophic factor delivery with a conduit can support, promote, and direct neurite outgrowth. EVAc polymers shaped into guidance channels that release neurotrophic factor demonstrated increased numbers of myelinated axons traversing an injury site relative to empty channels (E. G. Fine, et al., Eur. J. Neurosci. 15 (4) (2002) 589-601; F. M. Barras, et al., J. Neurosci. Res. 70 (6) (2002) 746-755; J. Bloch, et al., Exp. Neurol. 172 (2) (2001) 425-432.
In situations whereby it is desirous to deliver a gene encoding a particular growth factor to the site of injury to aid in growth of cells and tissues to replace those damaged by injury or disease, one general approach is to incorporate or attach the gene of interest directly into the polymeric matrix of the scaffold itself. While viral approaches can provide the most efficient means of gene transfer, biomaterials may be employed to enhance the efficacy of nonviral vectors by delaying clearance from the tissue, protecting against degradation, and maintaining effective concentrations for long times (Takakura, Y., Mahato, R. I., and Hashida, M. (1998), Adv. Drug Delivery Rev. 34: 93-108; Kawabata, K., Takakura, Y., and Hashida, M. (1995), Pharm. Res. 12: 825-830; Langer, R. (1998), Nature 392: 5-10; Pannier, A. K., and Shea, L. D. (2004), Mol. Ther. 10: 19-26.
Plasmid and modified nonviral vectors delivered from collagen scaffolds have been employed to promote tissue formation in several models, such as bone (Bonadio, J., Smiley, E., Patil, P., and Goldstein, S. (1999), Nat. Med. 5: 753-759; Fang, J., et al. (1996), Proc. Natl. Acad. Sci. USA 93: 5753-5758, cartilage (Pascher, A., et al. (2004), Gene Ther. 11: 133-141, nerve regeneration (Berry, M., et al. (2001), Mol. Cell Neurosci. 17: 706-716, and wound healing (Tyrone, J. W., et al. (2000), J. Surg. Res. 93: 230-236. Additionally, sustained delivery of plasmid DNA from a synthetic polymer, such as poly(lactide-co-glycolide) (PLG) matrices, significantly increased the level of in vivo transfection relative to direct injection (Shea, L. D., Smiley, E., Bonadio, J., and Mooney, D. J. (1999), Nat. Biotechnol. 17: 551-554). Transgene expression can influence tissue formation within or around the scaffold (Jang, J. H., Houchin, T. L., and Shea, L. D. (2004), Expert Rev. Med. Devices 1: 127-138. Although polymeric delivery of plasmid can promote gene transfer, the scaffold design parameters (e.g., porosity, loading) that regulate in vivo transgene expression are not well understood.
Accordingly, one object of the present invention is to provide a means of mechanical support for the attachment of cells to improve tissue regeneration. A second object is to incorporate into, or attach to, this biodegradable support either DNA encoding specific proteins, or the proteins themselves, such as growth factors or enzymes that can aid in modification of the microenvironment at the site of injury, thus allowing for more conducive conditions for cellular proliferation and tissue regeneration. Accordingly, the mechanisms for providing this means of tissue repair would be accomplished in a manner that can be applied at low cost and with great reproducibility.
The failure of others to achieve the above two objects is shown clearly by examples from the very fields that are most closely related to the purposes of the invention. These are the fields of tissue repair by providing a guidance tube or scaffold upon which new cells and tissue can be generated, while at the same time providing a means by which the new cells can incorporate genes of interest to aid in further cellular communication, proliferation and tissue repair.
All publications, patent applications, patents and other reference material mentioned are incorporated by reference in their entirety. In addition, the materials, methods and examples are only illustrative and are not intended to be limiting. The citation of references herein shall not be construed as an admission that such is prior art to the present invention.